1. Field Of The Invention
The present invention relates to methods for detecting the effects of cell affecting agents on living cells. Solutions or suspensions of cell affecting agents are flowed over cells and the effects of these agents are measured.
2. Description Of The Background Of The Invention
Studies of the effect of various cell affecting agents on living cells have been reported in the literature. See, e.g. Meisner, H. and Tenny, K. (1977) "pH as an indicator of free fatty acid release from adipocytes," J. Lipid Research, 18:774-776; Nilsson, N. and Belfrage, P. (1979) "Continuous monitoring of free fatty acid release from adipocytes by pH-stat titration," J. Lipid Research 20: 557-560; Reuss, L. , Weinman, S. and Constantin, J. (1984) "H.sup.+ and HCO.sub.3.sup.- transport at the apical membrane of the gallbladder epithelium," pp. 85-96 of Forte, J., Warnock, D. and Rector, F. Jr. (eds.) Hydrogen Ion Transport in Epithelia, Wiley-Interscience; Zeuthen, T. and Machen, T. (1984) "HCO.sub.3.sup.- /CO.sub.2 stimulates NA.sup.+ /H.sup.+ and Cl.sup.- /HCO.sub.3.sup.- exchange in Necturus gallbladder," pp. 97, 108 (ibid.); Handler, J. S. , Preston, A. S. and Steele , R. E. (1984) "Factors affecting the differentiation of epithelial transport and responsiveness to hormones," Federation Proceeding 43: 2221-2224; and Simmons, N. L., Brown, C. D. A. and Rugg, E. L. (1984) "The action of epinephrine on Madin-Darby canine kidney cells," Federation Proceedings, 43:2225-2229. These references disclose the detection of changes in pH and other electrical potentials by the addition of cell affecting agents to cells disposed in a relatively large amount of medium, i.e., a bulk medium. A disadvantage of these techniques is that the pH and other electrical potential measurements are taken from the bulk medium and do not necessarily reflect the actual values immediately adjacent to the cellular membranes of the living cells. Also, the high ratio of bulk volume to cell volume inevitably dilutes the effects of the cells on the properties of the extracellular medium. Accordingly, sensitivity is lost or greatly reduced.
Photoresponsive sensors for measuring biochemical systems are disclosed in various patent documents owned by the assignee of the present invention. See, e.g., U.S. Pat. Nos. 4,591,550 (Hafeman et al.) and 4,704,353 (Humphries et al.); and European Patent Application No. 213,825 (Hafeman et al.). U.S. Pat. No. 4,519,890 discloses a flow pH chamber. These patent publications disclose the use of microorganisms to measure changes in the environment of the solution to be measured. There is no disclosure in these publications of cells in micro flow chambers used to measure of the effects of cell affecting agents. See, also, U.S. Pat. Nos. 4, 737,464 (McConnell et al. ) and 4, 741,619 (Humphries et al. which are likewise owned by the assignee of the present invention.
Various ways of using fluorescence to measure extracellular effects of living cells and analytes are disclosed in the literature. e.g., Briggs et al. (1985) "Fiber Optic Probe Cytometer" J. Immunological Methods, 81:73-81; Hafeman et al. (1984) "Superoxide Enhances Photo Bleaching During Cellular Immune Attack Against Fluorescent Lipid Monolayer Membranes" Biochemica Biophysica Acta 772:20-28; and U.S. Pat. Nos. 4,560,881 and 4,564,598.